During my postdoctoral fellowship, I successfully purified and identified the first mammalian N-terminal methyltransferase, NRMT. Shown by microarray analysis to have reduced expression/copy number in human breast cancers, I have also illustrated NRMT knockdown results in increased cell proliferation and a multi- spindle phenotype. Given that NRMT knockdown results in these two cancer prone phenotypes, I intend to further study how it is regulated, how N-terminal methylation affects substrate function, and how NRMT misregulation leads to tumorigenesis. My goal after completion of my postdoctoral work is to obtain a tenure- track position at an academic research institution and establish an independent research program. Successful completion of my research goals includes training in a mouse mammary transplant model. As I have no previous mouse research experience, the additional training time provided by a K99 will be imperative for learning these skills. Obtaining a K99, will also allow for further training in mass spectrometry, fluorescent- activating cell sorting, and FRET analysis. The long-term goal of my research is to use the NRMT mouse model to dissect the signaling pathways that lead to breast cancer. As personalized medicine treatments become increasingly necessary, I want to create a model where combinations of genes can be easily manipulated simultaneously. These models can be used to test which current drug treatments are most effective for patients with specific combinations of mutations and to screen for new and more effective therapeutic agents. Research The list of genes involved in tumorigenesis is quite extensive, however, many of their biological functions remain unknown. One such gene, Mettl11a (now renamed NRMT), has been shown to be under-expressed in breast cancers, but has only recently been identified as the first mammalian N-terminal methyltransferase. Reduction of NRMT also results in a multi-spindle phenotype, and the associated aneuploidy is often considered to contribute to cancer progression. As NRMT is a newly discovered protein, the goals of this proposal are to understand the role of N-terminal methylation on protein and cellular function, in order to study how its misregulation leads to tumorigenesis. The first two aims, determining whether N-terminal methylation is constitutive and determining whether N-terminal methylation universally alters the DNA binding of its substrates, are designed to better understand the basic biology of N-terminal methylation. These aims will involve mass spectrometry, fluorescent-activating cell sorting, FRET analysis, and construction of a knockout mouse. The objective of the third aim is to determine the role of NRMT in development and tumorigenesis. A mouse mammary transplant model will be established for assaying if the multi-spindle NRMT knockdown phenotype leads to developmental defects and/or tumorigenesis. The key element of the career development aspect of this proposal will be learning the mouse system needed for successful completion of the third aim. The experiments of aims one and two have been designed for the downtime between surgeries and gland or tumor harvesting. Environment The University of Virginia is well equipped for completion of all three aims of this proposal. The on-campus mass spectrometry facility, of advisory committee member Dr. Don Hunt, has a Fourier transform mass spectrometer necessary for distinguishing N-terminal methylation from acetylation. The UVA Flow Cytometry Core will provide the training necessary for all fluorescent-activating cell sorting experiments. The UVA Gene Targeting and Transgenic Facility will create the NRMT knockout mouse. The Research Histology Core will paraffin embed and make slides of all normal mouse tissue and tumors. My advisory committee member Dr. Amy Bouton will aid in interpretation of the histology. The expertise and reagents of advisory committee member Dr. Todd Stukenberg will aid in characterization of the NRMT multi-spindle phenotype. The UVA W.M. Keck Center for Cellular Imaging supplies state-of-the art imaging facilities for immunofluorescent imaging of mouse tissue samples and training in FRET analysis. The Macara lab currently has three expert mouse biologists available for training in mouse handling and surgical techniques. In addition, UVA offers numerous courses aimed at career development/training, including the UVA transgenic methods and applications workshop, the Flow Cytometry Training Workshop, the Workshop on FRET Microscopy, and a monthly career development seminar series offered by the Office of Postdoctoral Professional Development. PUBLIC HEALTH RELEVANCE: The biological roles of many genes involved in tumorigenesis remain unknown. We need to study their functions in order to achieve a complete understanding of cancer progression and obtain an inclusive list of potential druggable targets. One such protein, NRMT, the newly identified mammalian N-terminal methyltransferase, is under-expressed in breast cancers, and its knockdown results in mitotic defects often considered to contribute to cancer progression. The goal of this proposal is to understand how NRMT is regulated and how its misregulation contributes to tumorigenesis. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" above summarizes the final opinions of the committee.